In this project, we will test new compounds that inhibit transcription of human telomerase reverse transcriptase (hTERT) through specific binding of G-quadruplexes (GQs) formed in the hTERT promoter. Over-expressed in cancer cells, telomerase elongates telomere and avoids programmed cell death (apoptosis). Current telomerase targeting approaches have demonstrated efficacy to kill cancer cells, which results in several drugs being tested in clinical trials. However, these approaches directly inhibit the canonical activity of telomerase, leading to the shortening of telomere that kills cancer cells only after a long lag time that is not suitable for therapeutic applications. An emerging strategy in the field is to target the biogenesis of telomerase. Our recent finding has indicated that by inhibition of hTERT transcription, cancer cells are killed within days through interfering with non-canonical telomerase activities that reduce oxidative stress in mitochondria and avoid apoptosis. While the presence of G-quadruplex (GQ) in human cells has been proven, bioinformatics have revealed the enrichment of GQ hosting sequences in promoter regions of many oncogenes. In the hTERT core promoter, our labs have found that formation of two tandem GQs can inhibit telomerase transcription. Mutations identified in melanoma patients are located in this region, resulting in compromised GQ structures and loss of transcriptional silencing. Given that telomerase is overexpressed in melanoma patients, it provides strong evidence that the formation of GQs in the hTERT promoter inhibits telomerase expression. We have also found that small molecules facilitating the folding of these silencer GQs can decrease telomerase activity, which corroborates the link between GQ formation in hTERT promoter and reduced telomerase production. Specificity presents a major hurdle to inhibit telomerase via G-quadruplex targeting in hTERT promoter. In human genome, over 716,000 sites can form GQs. Due to the generic nature of aromatic stacking and electrostatic interactions employed in GQ-binding ligands, current small molecules do not recognize specific quadruplexes, which brings side effects. Since hTERT promoter GQs are flanked by duplex DNA regions, we will covalently conjugate quadruplex-binding ligands, such as pyridostatin, to duplex DNA recognition elements, such as polyamides. While the dual targeting increases binding affinity, selective recognition of neighboring duplex DNA offers specificity for the G-quadruplexes in the hTERT promoter. We will use single- molecule mechanoanalytical approaches to evaluate the specificity and affinity of these new compounds to hTERT GQs. In these methods, the potency of a ligand is measured by the mechanical stability of ligand- bound G-quadruplexes, which serve as mechanical blocks to motor proteins such as RNA polymerase. These single-molecule results will be further validated by complementary biochemical and cellular assays including luciferase, transcription/translation, and telomerase assays on Taxotere-resistant prostate cancer cell lines.